Abstract
Variations in the potential glycosylation sites were observed in hemagglutinin (HA) sequences of H9N2 avian influenza virus isolated in China, deposited in the Influenza Virus Resource of NCBI before 2017, which showed a deleted glycosylation site at amino acid residue 218, and an introduced glycosylation site at amino acid residue 313. Based on the variations in the glycosylation sites at these amino acids, H9N2 avian influenza viruses could be divided into three categories. Firstly, most of the H9N2 influenza viruses were 218G+ viruses; less 313G+ viruses were isolated between 1997 and 2004. Secondly, the occurrence of the 218G+/313G+ viruses increased, while the 218G+/313G− viruses decreased from 2005 to 2012. Thirdly, from 2013 to 2016, the 218G−/313G+ viruses were predominant compared to the 218G+/313G+ viruses. Here, based on an F/98 virus backbone, a 218G+/313G− virus, two reassortment viruses were generated, and named rF/HA218G+/313G+ and rF/HA 218G+/313G−, respectively. HA protein migration demonstrated that the glycosylation sites at amino acid residues 313 and 218 were both functional. The absence of the glycosylation site at amino acid residue 218 and the presence of the glycosylation site at amino acid residue 313 increased antibody binding and moderately prevented the virus from escaping neutralization with homologous antisera. Additionally, compared to the F/98 virus (218G+/313G−), the viruses rF/HA218G+/313G+ or rF/HA218G−/313G+ showed significantly increased infectivity of MDCK cells, chicken embryo eggs, and trachea and lung tissue of SPF chickens, but did not display differences in airborne spread in chickens or infectivity of mice compared with its parental virus F/98.
Highlights
The H9N2 avian influenza virus was first detected in the North American turkey in 1966, after which it spread throughout the world, causing huge economic losses in the poultry industry
To understand the function of these variations in NGL sites between the amino acids 218 and 313 in the HA of the H9N2 influ‐ enza viruses last decade, two reassortant viruses, rF/ HA218G+/313G+ and rF/HA218G−/313G+, were gener‐ ated based on the F98 virus backbone
The results of the HI assay showed that the two reassortant viruses, rF/HA218G+/313G+ and rF/ HA218G−/313G+, viruses only reacted with F/98 serum, yet did not react with anti-NDV (Newcastle disease virus) and anti-H5 sera (Table 1)
Summary
The H9N2 avian influenza virus was first detected in the North American turkey in 1966, after which it spread throughout the world, causing huge economic losses in the poultry industry. The NLG status of the receptor-binding domain of HA in IAV mediates protective antibody responses against the 1918 and 2009 pandemic H1N1 viruses [11, 12]. HA protein of IAV is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection [1, 8, 16]. The variations in the glycosylation (abbreviates G) sites at amino acid residues 218 (named 218G+, Asn-Arg-Thr-Phe, NRTF) and 313 (named 313G+, Asn-Cys-Ser-Lys, NCSK) emerged in the process of the evolution of H9N2 avian influenza viruses. The effect of the variations in NLG sites on the H9N2 influenza virus was explored in antibody binding, and infectivity of embryonated eggs of specific-pathogen-free (SPF), MDCK cells, or SPF chickens
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